Biol Trace Elem Res (2018) 182:339–353

DOI 10.1007/s12011-017-1092-1

Effect of Dietary Phytase Supplementation on Bone and Hyaline

Cartilage Development of Broilers Fed with Organically
Complexed Copper in a Cu-Deficient Diet
Siemowit Muszyński 1 & Ewa Tomaszewska 2 & Małgorzata Kwiecień 3 &
Piotr Dobrowolski 4 & Agnieszka Tomczyk 2

Received: 15 April 2017 / Accepted: 28 June 2017 / Published online: 15 July 2017
# The Author(s) 2017. This article is an open access publication

Abstract Tibial mechanical, chemical, and
histomorphometrical traits were investigated for growing
male Ross 308 broiler chickens fed diets that had copper
(Cu) from organic source at a lowered level of 25% of the
daily requirement (4 mg kg−1 of a premix) with or without
phytase. Dietary treatments were control non-copper, non-
phytase group (0 Suppl); 4 mg kg−1 Cu non-phytase group
(25%Cu); and 4 mg kg−1 Cu + 500 FTU kg−1 phytase group
(25%Cu + phyt). The results show that birds fed with the
addition of phytase exhibited improved weight gain and final
body weight and had increased serum IGF-1 and osteocalcin
concentrations. The serum concentration of Cu and P did not
differ between groups; however, Ca concentration decreased
in the 25%Cu + phyt group when compared to the 25%Cu
group. Added Cu increased bone Ca, P, Cu, and ash content
in Cu-supplemented groups, but bone weight and length in-
creased only by the addition of phytase. Bone geometry,
yield, and ultimate strengths were affected by Cu and phytase
addition. A decrease of the elastic stress and ultimate stress of
the tibia in Cu-supplemented groups was observed. The
* Siemowit Muszyński
siemowit.muszynski@up.lublin.pl
1
Department of Physics, Faculty of Production Engineering,
University of Life Sciences in Lublin, Akademicka 13,
20-950 Lublin, Poland
2
Department of Animal Physiology, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, Akademicka 12,
20-950 Lublin, Poland
3
Institute of Animal Nutrition and Bromatology, University of Life
Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
4
Department of Comparative Anatomy and Anthropology, Maria
Curie-Skłodowska University, Akademicka 19,
20-033 Lublin, Poland
histomorphometric analysis showed a positive effect of Cu
supplementation on real bone volume and trabecular thick-
ness in the tibia metaphyseal trabeculae; additionally, phytase
increased the trabeculea number. The supplementation with
Cu significantly increased the total articular cartilage and
growth plate cartilage thickness; however, the changes in
thickness of particular zones were dependent upon phytase
addition. In summary, dietary Cu supplements given to grow-
ing broilers with Cu in their diet restricted to 25% of the daily
requirement had a positive effect on bone metabolism, and
phytase supplementation additionally improved cartilage
development.
Keywords Organic copper . Phytase . Bone
histomorphometry . Mechanical testing . Broiler chicken
Introduction
Copper (Cu) as an essential trace element has many physio-
logical functions in animals and humans, including skeletal
mineralization, erythropoiesis, leukopoiesis, connective tissue
synthesis, myelin formation, melanin pigment synthesis, cat-
echolamine metabolism, thermal regulation, cholesterol me-
tabolism, immune function, cardiac function, and glucose
metabolic regulation [1, 2]. It is known that Cu takes part in
bone metabolism as an essential co-factor needed for the ac-
tion of lysyl oxidase [3]. This Cu-dependent enzyme initiates
the critical process of covalent cross-linkage formation in elas-
tin and collagen in bones and other connective tissues [1, 4].
Inhibition of lysyl oxidase enzyme activity results in
osteolathyrism and negatively affects bone and cartilage for-
mation [5]. Furthermore, studies on Cu supplementation indi-
cate that Cu deficiency leads to bone loss (osteopenia or oste-
oporosis), demineralization, a failure of ossification of growth
340
centers, and neuropenia [1, 2]. It is caused mainly by the
decreased function of osteoblasts (bone tissue-forming cells)
because the action of osteoclasts (bone tissue-resorbing cells)
remains unaffected [3]. All of these phenomena lead to the
decrease in bone mechanical strength and result in consequent
fractures [4, 6].
Trace minerals are essential in the diets of poultry because
they participate in the biochemical processes required for the
maintenance of normal growth and development, including
bone and eggshell formation and development of the avian
embryo [7]. Moreover, in poultry, there is substantial interest
in using Cu as a health promoter as an alternative to antibiotics
that can produce equivalent effects on chicken performance
[8]. Copper is often added to poultry diets at prophylactic
concentrations for its growth-promoting effects by influencing
the microbial populations within the gastrointestinal tract [9].
The Cu requirement in the diets of chicken has been
established to be 8 mg kg−1 [10], but a greater amount of Cu
than is required has often been included in commercial broiler
diet. The reason for this is that the differences in the concen-
trations of Cu and its bioavailability in various feed ingredi-
ents are as a result of the variations in processing procedures
and in the cultivating conditions used for feed ingredients
[10]. Therefore, several supplemental Cu sources such as in-
organic sources (copper sulfate or carbonate) and organic
sources (chelated form with higher Cu bioavailability) have
been added to livestock diet, including broilers [6, 11–13].
Chelates are reported to have significantly higher absorption
rates from the intestine due to their unique chemical structure
as compared to inorganic salts, and it has been shown that
supplementation with 4 mg kg−1 of Cu from organic sources
may be sufficient for normal broiler growth [11]. Furthermore,
organically complexed trace minerals make available alterna-
tive pathways for absorption, thus leading to a reduction in the
excretion of minerals to the environment [8, 11]. Therefore, it
seems that the use of a mineral chelate allows for maintaining
performance while concomitantly reducing the mineral con-
tent in manure [13, 14].
The skeletal system consists of bones, cartilage, ligaments,
and tendons and accounts for about 20% of the body weight of
most vertebrates. It has been demonstrated that the use of Cu
in the form of glycinate compounds at much reduced amounts
as opposed to using sulfate at its recommended dose does not
cause deterioration in the physical, mechanical, and morpho-
metric properties of femur in chickens [2, 14, 15]. Thus, with
regard to the important role of Cu in the development of
bones, it is hypothesized that the use of Cu in the more digest-
ible and assimilable form of chelate might improve the growth
and development of the skeletal system in broiler chickens.
Phytic acid is a naturally occurring organic complex found
in plants. About 70% of P present in the feedstuffs of plant
origin used in the feed for poultry is in phytate form, inacces-
sible to non-ruminant animals [14]. Phytic acid, as a reactive
Muszyński et al.
anion, forms insoluble salts with Cu and other divalent cations
[14]. These complexes are indigestible for poultry, and the
utilization of microelements bound in phytate form is lowered
or impossible [14]. However, it is well known that the use of
phytase, an enzyme that hydrolyzes phytate complexes, in-
creases the availability of trace minerals [16, 17]. Studies to
quantify the bioavailability of dietary Cu given at marginally
deficient amounts from alternative sources of Cu are numer-
ous [2, 6, 11]. However, to date, the relationship between the
dietary status of Cu and the presence of phytase on bone
health and cartilage development has not been well studied,
including on broiler chickens.
With regards to the important role of Cu in bone metabolism
during development, the objective of this study was to compare
the effects of administrating amino acid Cu complex in grow-
ing male Ross 308 chickens fed a Cu-poor diet, which ensured
higher Cu bioavailability through enhanced absorption from
the intestine, to those fed the same diet but which were addi-
tionally enriched with phytase. This was assessed on the basis
of the mechanical, geometric, and histomorphometric parame-
ters of the tibia, bone Cu content, and determination of the
concentration of hormones of the somatotropic axis in serum.
It is hypothesized that the use of Cu in the more assailable form
of glycinate chelate might improve the development of the
skeletal system in broiler chickens, even if it is administered
at the reduced level of 25% of the daily recommended dose.
Thus, to evaluate the possible response of broilers to organical-
ly complexed Cu and the effect of phytase inclusion into the
diet, the present study was conducted with a control diet devoid
of both phytase and mineral supplementation of Cu in a trace
mineral premix.
Materials and Methods
The experimental procedures used throughout this study were
approved by the Local Ethics Committee on Animal
Experimentation of the University of Life Sciences of
Lublin, Poland. The birds were maintained in an animal house
according to the guidelines of this committee. All efforts were
made to minimize the number of animals used as well as their
suffering.
Animals and Experimental Design
A total of 120 1-day-old Ross 308 broiler chickens were ob-
tained from a commercial hatchery. The birds were weighed
after hatching and randomly selected to one of three dietary
treatments, each group containing 40 chickens. The chickens
were assigned to either a control group (the 0 Suppl group; 40
birds divided into 10 pens with 4 birds per pen), or a group fed
with lowered level of organic Cu in the form of glycinate
chelate (Cu-Gly) as experimental group I (the Cu25% group;
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
40 birds divided into 10 pens with 4 birds in each pen), or a
group fed with lowered level of organic Cu in the form of
glycinate chelate simultaneously enriched with phytase as ex-
perimental group II (the Cu25% + phyt group; 40 birds divid-
ed into 10 pens with 4 birds in each pen). All birds were raised
in battery cages (76 × 97 × 45 cm, width × length × height)
placed in an environmentally controlled room and kept under
standard rearing conditions and air temperature set at the op-
timal level depending on age. During the first week, the
chickens were kept at 33 °C, which was reduced by 2 °C
weekly, until the final temperature of 24 °C. The chickens
had constant access to fresh water and appropriate feed sup-
plied ad libitum in accordance with this stage of the produc-
tion cycle (Table 1). To evaluate the growth rate, the birds’
daily body weight gains were recorded. The birds were fed a
diet corresponding to the periods of rearing: starter (1–
21 days), grower (22–35 days), and finisher (36–42 days).
The chickens received a starter diet in the form of crumble,
and grower and finisher diets in the form of pellets. At the end
of the experiment, 10 birds randomly selected from the control
(1 bird from each pen) and experimental groups I and II (1 bird
from each pen) were weighed and slaughtered by cutting the
carotid arteries. Ten hours before the slaughter, the selected
birds were not given feed, but only provided with unlimited
access to water.
Immediately after slaughter, the tibiae were dissected and
cleaned from the remnants of adherent tissues and their weight
and length measured. Directly after the measurements, each
bone was wrapped in gauze soaked in isotonic saline and
frozen at −25 °C for further analyses.
Supplementation of Cu Amino Acid Chelate and Phytase
The control group (the 0 Suppl group) was fed basal diet
supplemented with the premix which did not provide external
Cu (0 mg kg−1). The experimental diets were formulated by
supplementing a corn-wheat-soyabean meal mixture (Table 1)
with lowered levels (25% of the total daily recommended
amount for Ross 308 broiler, 4 mg kg−1) of Cu from Gly-
Cu, with or without phytase (500 FTU kg−1). The experiment
involved the use of Glystar Forte chelate (Arkop Sp. z o.o.,
Bukowno, Poland) containing 16% of Cu and Ronozyme®
HiPhos 6-phytase (DSM Nutritional Products, Mszczonów,
Poland) produced by a genetically modified strain of
Aspergillus oryzae. Application of glycine chelate was in ac-
cordance with the EU Directive 1334/2003 [18].
The basal corn-wheat-soybean meal diet (Table 1) contain-
ing (by analysis) 6.1 mg kg−1 (starter), 6.21 mg kg −1 (grower),
and 5.91 mg kg−1 (finisher) of Cu from plants as the feed basis
was formulated to meet or exceed nutritional requirements
[10]. The amount of Cu in the premix was based on nutritional
recommendations for Ross 308 broilers [10, 19], i.e.,
16 mg kg −1 of Cu, irrespective of its content in the
341
components of the basal diet. According to these recommen-
dations, the Cu content should be the same in all periods of
rearing, which was taken into account in the study [10, 19].
The nutrient composition of the basal diet was analyzed
using standard methods: total phosphorus, calorimetrically with
a Helios Alpha UV-VIS apparatus (Spectronic Unicam, Leeds,
UK) [20], phytic phosphorus, by the Frühbeck et al. method
[21], and the Cu, Fe, and Ca content in feed samples deter-
mined after ashing at 550 °C using the AAS flame technique
in a Unicam 939 AA Spectrometer (Shimadzu Corp., Tokyo,
Japan) apparatus, according to the methods of AOAC [20].
The amino acid composition in the diet was determined by
ion exchange chromatography using an INGOS AAA 400
amino acid analyzer with post-column derivatization of nin-
hydrin and spectrophotometric detection [20]. Cysteine and
methionine (sulfur amino acids) were determined in a separate
analysis as described previously [15], according to the method
of AOAC [20]. Assimilable lysine was determined based on
the difference between total lysine and the so-called residual
lysine which did not react with DNFB (dinitrofluorobenzene)
[15]. Following this reaction, the tested samples were again
subjected to acid hydrolysis [22].
Serum Biochemical Analyses
Each chicken was fasted for 12 h before blood collection. The
blood was collected using standard venipuncture from the
brachial vein; next, after clotting at room temperature, it was
centrifuged and frozen at −80 °C for further analysis. The
blood serum concentrations of copper, calcium, and phospho-
rus were determined by a colorimetric method using a
Metrolab 2300 GL unit (Metrolab SA, Argentina) and sets
of biochemical reagents produced by BioMaxima (Lublin,
Poland) [20].
Growth Hormone and Bone Turnover Markers
The serum concentration of chicken growth hormone, insulin-
like growth factor 1 (IGF-1), osteocalcin, and leptin were de-
termined using an enzyme-linked immunosorbent assay kit
(ELISA; Uscn Life Science Inc. Wuhan, China) with mini-
mum detectable concentrations of 0.056 ng ml − 1 ,
7.4 pg ml−1, 0.67 pg ml−1, and 14.8 pg ml−1, respectively.
Mechanical Properties
The mechanical properties of the tibia were determined for all
the groups after 3-h thawing at room temperature using the
three-point bending test of bone mid-diaphysis. The mechani-
cal properties were examined on a Zwick Z010 universal test-
ing machine (Zwick GmbH & Co. KG, Ulm, Germany),
equipped with a measuring head of operation range up to
10 kN, linked to a computer with testXpert II 3.1 software
342 Muszyński et al.

Table 1 Composition and

nutritive value of the Ingredients (%) Starter Grower Finisher
experimental diet (1–21 days) (22–35 days) (36–42 days)

Corn 24.5 40.0 40.0

Wheat 42.9 27.9 28.8
Soybean mealf 25.0 24.9 22.9
Soybean oil 2.50 3.69 3.98
Phosphate 1—Ca 0.90 0.90 0.81
Fodder chalk 1.40 1.13 1.09
Sodium bicarbonate 0.08 0.08 0.08
NaCl 0.29 0.25 0.26
Premix vitamin (no Cu) 0.50a 0.50b 0.50c
Concentrate protein—fattyg 1.00 – 1.00
DL-Methionine 99% 0.30 0.23 0.23
L-Lysine HCl 0.42 0.28 0.27
L-Threonine 99% 0.18 0.13 0.07
The nutritional value of diet
ME (MJ kg−1)e 12.7 13.1 13.2
Total protein (%)d 21.2 20.4 19.9
Crude fiber (%)d 1.64 1.59 1.73
Crude fat (%)d 4.57 5.42 5.53
Lysine, total (%)d 1.28 1.14 1.08
Met + Cys (%)d 0.92 0.81 0.82
Total Ca (%)d 0.87 0.79 0.76
Total P (%)d 0.65 0.66 0.64
Phytate P (%)d 0.32 0.32 0.32
Bioavailable P (%)e 0.42 0.41 0.39
Total Ca/P bioavailablee 2.12 1.90 1.92
Cu from plants in basal diet (mg kg−1)d 6.04 6.07 5.91
Fe (mg kg−1)d 40.31 39.82 38.61
Cu (mg kg−1)d
4 mg Cu-Gly 10.28 10.34 10.21
4 mg Cu-Gly + Phyt 11.12 10.87 11.01
a
Content of vitamins and minerals per 1 kg of starter: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 15000 UI, vitamin D3 5000 UI, vitamin E 75 mg, vitamin K3 4 mg, vitamin B1 3 mg, vitamin B2 8 mg,
vitamin B6 5 mg, vitamin B12 0.016 mg, biotin 0.2 mg, folic acid 2 mg, nicotinic acid 60 mg, pantothenic acid
18 mg, choline 1800 mg
b
Content of vitamins and minerals per 1 kg of grower: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 12000 UI, vitamin D3 5000 UI, vitamin E 50 mg, vitamin K3 3 mg, vitamin B1 2 mg, vitamin B2 6 mg,
vitamin B6 4 mg, vitamin B12 0.016 μg, biotin 0.2 mg, folic acid 1.75 mg, nicotinic acid 60 mg, pantothenic acid
18 mg, choline 1600 mg
c
Content of vitamins and minerals per 1 kg of finisher: Mn 100 mg, I 1 mg, Fe 40 mg, Cu 16 mg, Se 0.15 mg,
vitamin A 12000 UI, vitamin D3 5000 UI, vitamin E 50 mg, vitamin K3 2 mg, vitamin B1 2 mg, vitamin B2 5 mg,
vitamin B6 3 mg, vitamin B12 0.011 μg, biotin 0.05 mg, folic acid 1.5 mg, nicotinic acid 35 mg, pantothenic acid
18 mg, choline 1600 mg
d
Analyzed values
e
Calculated values
f
Forty-six percent total protein in dry matter
g
One kilogram concentrate protein-fatty contains 2% crude fat, 39% crude protein, 10.8 MJ ME

(Zwick GmbH & Co. KG, Ulm, Germany), registering the between the supports was set at 40% of the total bone length.
relationship between force perpendicular to the longitudinal The measuring head loaded bone samples with a constant
axis of the bone and the resulting displacement. The distance speed of 10 mm min−1 until fracture [23]. The ultimate load
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
was determined as the force causing bone fracture and the yield
load as maximal force under an elastic (reversible) deformation
of the bone [24]. Moreover, on the basis of measured geometric
and mechanical traits, the material properties of the mid-
diaphyseal fragment of the bone were calculated. These traits
describe the specific mechanical properties of the midshaft cor-
tical tissue and are independent of the bone size and the condi-
tions under which the strength tests were conducted. The bend-
ing moment can be described as a yield load adjusted to the
bone length, and it indicates the bone elastic load capability
[25]. The elastic stress reflects the elastic strength of midshaft
cortical bone; the ultimate stress is equal to the maximum stress
a bone can withstand in bending before fracture [25].
Geometric Parameters
The geometric properties such as the cross section area (A), the
mean relative wall thickness (MRWT), and the cortical index
(CI; defined as the ratio of the thickness of the cortical part to
the thickness of the midshaft measured at the middle part of the
bone) were estimated on the basis of the horizontal and vertical
diameter measurements of the mid-diaphyseal cross section of
the bone with the previously described method [26]. Moreover,
as during the strength analysis the bone was loaded in the A-P
plane, the second (cross-sectional) moment of inertia Ix and the
radius of gyration Rg about the medial-lateral (M-L) axis were
calculated [25]. The second moment of inertia Ix is not a direct
bone geometric trait, but is a critical property in terms of the
bone bending rigidity evaluation.
The samples of the proximal end of each bone were sub-
jected to histology as described previously [2]. The site and
size (approximately 3 mm in length of both analyzed carti-
lages) of the areas of interest which were measured were cho-
sen on the basis of motoric properties of the body—the knee
joint in particular—as was described previously [27]. Two
methods of staining were used: the Goldner’s trichrome to
assess the morphology of the growth plate and articular carti-
lage, and the safranin-O staining to visualize the cartilage
proteoglycans [28]. Briefly, the sagittal sections through the
middle of the lateral condyle of each tibia were cut strictly
according to the previously described method and equipment
[27]. Safranin-O staining was applied to the visual assessment
of Mankin’s histological and histochemical grading system for
evaluation of the articular cartilage [29, 30].
The thickness of the following zones: reserve (I), prolifer-
ation (II), hypertrophy (III), and ossification (IV) were mea-
sured at four sites along the growth plate cartilage, and an
average was calculated as described previously [30].
Similarly, the thickness of the main zones of the articular
cartilage, i.e., horizontal (superficial surface, I), transitional
(II), and radial (III), was measured as described previously [2].
The Picrosirius red staining (PSR) was employed to assess
the morphology of the articular cartilage and to evaluate the
343
distribution of thick (mature) and thin (immature) collagen
fibers in the articular cartilage [31–33]. The sections stained
with PSR were analyzed using a Leica DM 2500 microscope
(Leica Microsystems, Wetzlar, Germany) equipped with fil-
ters to provide circularly polarized illumination. Images were
documented by a high-resolution digital camera (Leica
Microsystems, Wetzlar, Germany).
The bone volume (BV), tissue volume (TV), relative bone
volume (BV/TV), trabecular thickness (Tb.Th), trabecular
separation (Tb.Sp), trabecular number (Tb.N), and fractal di-
mension (Fd) of the trabecular bone were measured as de-
scribed previously [28].
Ca, P, and Ash Content in Bone
After evaluating the strength and structural properties, the
bones were defatted, dried at 105 °C to a constant mass, and
finally mineralized in a muffle furnace at 500 °C [2, 22]. The
content of the mineral components (Ca, P, Cu) of the bones
was determined by an atomic absorption spectrometry using a
Unicam 939/959 apparatus [20]. The percentage of bone ash
and the content of Ca, P, and Cu in the bone were calculated as
part of components from the crude ash.
Statistical Analysis
All results are expressed as mean ± SD (standard devia-
tion). The differences between the means were tested with
one-way ANOVA and post hoc Tukey’s HSD test as the
correction for multiple comparisons. Normal distribution of
data was examined using the Shapiro-Wilk W-test, and
equality of variance was tested by the Brown-Forsythe
test. A P value of less than 0.05 was considered statisti-
cally significant. All statistical analyses were carried out
by means of Statistica 12 software (StatSoft, Inc., Tulsa,
OK, USA; http://www.statsoft.com).
Results
Body Weight
The initial body weights of the control and birds treated with
the organic Cu form (regardless of the presence of phytase)
were similar (Table 2). At the end of the experiment, chickens
fed Cu-poor diet supplemented with the phytase (the
Cu25% + phyt group) weighed significantly more than the
birds from the control group (the 0 Suppl group) and the
Cu25% group (Table 2). Daily weight gain was also signifi-
cantly higher in the Cu25% + phyt group compared to the
control and the Cu25% groups (Table 2).
344 Muszyński et al.

Table 2 The body weight (initial

and 42-day-old as final body Group Number Body weight (g) Daily weight gain (g)
weight) of control broilers and
supplemented with Cu in 25% of Initial body weight Final body weight
daily demand dependently on
phytase supplementation 0 Suppl 10 44.1 ± 1.0 2262 ± 127a 49.4 ± 4.3a
Cu25% 10 43.8 ± 1.0 2281 ± 144a 53.3 ± 3.4a
Cu25% + phyt 10 44.0 ± 0.8 2716 ± 160b 63.6 ± 3.8b
SEM 0.17 46 1.31
P value P = 0.775 P < 0.001 P < 0.001

Mean values in columns with different lowercase letters differ significantly at P < 0.05; data given are mean ± SD
(standard deviation)
SEM standard error of the mean, 0 Suppl the control group without received Cu in premix, Cu25% the group
received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the group received Cu in 25% of daily demand
from Cu-Gly with phytase

The Content of Ca, Total P, and Cu in Blood Serum The Content of P, Ca, and Cu in Bone

The Cu serum concentration of the birds from both experi-
mental groups (irrespective of the phytase supplementation)
reached similar values as those in the control group (Fig. 1).
Similarly, total P serum concentration was similar in the con-
trol chickens and birds supplemented with the Cu-Gly, regard-
less of the phytase addition (Fig. 1). The Ca serum concentra-
tion of the chickens from the Cu25% + phyt group was lower
compared to the control values noted in chickens from the
Cu25% group (Fig. 1). However, the serum Ca/P ratio did
not differ between groups (Fig. 1).
The bone P, Ca, and bone crude ash content in the
chickens supplemented with Cu in the Cu-Gly form
in the Cu-poor diet, irrespective of the phytase pres-
ence, was higher compared to the control values
reached in the 0 Suppl group (Fig. 2). However, the
bone Ca/P ratio did not differ between groups (Fig. 2).
The bone Cu content in both groups supplemented
with Cu in 25% of daily demand was higher compared
to the control chickens which were fed the Cu-
deprived diet (Fig. 2).

Fig. 1 The serum concentration

of calcium (Ca), copper (Cu),
phosphorus (P), and Ca/P ratio in
42-day-old chickens treated with
Cu in organic (Cu-Gly) form
dependently on phytase
supplementation. Data given are
mean ± SD. a, b Values with
different letters differ significantly
at P < 0.05. 0 Suppl—the control
group without received Cu in
premix. Cu25%—the group
received Cu in 25% of daily
demand from Cu-Gly. Cu25% +
phyt—the group received Cu in
25% of daily demand from Cu-
Gly with phytase
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
Fig. 2 The bone content of calcium (Ca), phosphorus (P), copper (Cu),
crude ash, and bone Ca/P ratio in 42-day-old chickens treated with Cu in
organic (Cu-Gly) form dependently on phytase supplementation. Data
Bone Morphology, Geometry, and Mechanical Properties
The intake of Cu in the Cu-Gly form at the concentration of
25% of daily requirement with the addition of phytase in-
creased the bone weight, length, and weight/length ratio
compared to the other groups (Table 3). Moreover, Cu-Gly
administration at 25% of daily requirement significantly al-
tered all measured diameters; however, the greatest increases
were noted in the group additionally supplemented with
phytase (Table 3). As a result, the chickens fed the Cu-
poor diet irrespective of the phytase addition had an in-
creased cross section area as compared to the control
chickens. However, the mean relative wall thickness de-
creased in the Cu25% group, while the cortical index in-
creased in the Cu25% + phyt group compared to the control
0 Suppl group (Table 3). The change of the cross section
dimensions of the midshaft resulted in a significant change
of the values of the midshaft volume and the cross-sectional
moment of inertia in both groups fed the Cu-poor diet (with
the highest values in the group with phytase addition) when
compared to control birds fed the Cu-deprived diet
(Table 3). Similarly, an increase in the radius of gyration
of both experimental groups was observed.
The addition of phytase in the Cu-poor diet resulted in an
increase in the yield strength, the ultimate strength, and the
bending moment compared to the control diet (Cu-deprived)
and the Cu-poor diet without the phytase. However, for both
experimental groups, the Cu supplementation led to a signif-
icant decrease of the elastic stress and the ultimate stress of the
midshaft cortical bone (Table 3).
345
given are mean ± SD. a, b Values with different letters differ significantly
at P < 0.05. The description of the groups as in Fig. 1
Bone Histomorphometry
The microscopic assessment of cancellous bone in both ex-
perimental groups supplemented with Cu-Gly at the concen-
tration of 25% of the daily requirement showed a significant
increase in the real bone volume (BV/TV), the mean (Tb.Th
mean), and the maximal (Tb.Th max) trabecular thickness
(Table 4). However, an increase in the mean (Tb.Sp mean)
and the maximal (Tb.Sp max) trabecular space in the
Cu25% group when compared to the control group was noted
while the phytase addition resulted in a decrease of the Tb.Sp
mean and the Tb.Sp max (Table 4). Moreover, in the
Cu25% + phyt group, an increase in the trabecula number
(Tb.N) was observed. The fractal dimension (Fd) of the tra-
becular bone increased significantly in the Cu25% + phyt
group and decreased in the Cu25% group when compared to
the control group.
Morphology of the Articular and Growth Plate Cartilages
The examined joints had no visible lesions or degener-
ative changes. The supplementation of Cu-Gly to the
feed of growing chickens (regardless of phytase addi-
tion) significantly increased the total, I, and II zones
of the articular cartilage, while the greatest change was
observed in the group fed with the phytase additive
(Fig. 3). Zone III became wider in the Cu25% group
and narrowed in the Cu25% + phyt group when com-
pared to the control group. As a result, the articular
cartilage total thickness increased in both experimental
346 Muszyński et al.

Table 3 The physical,

mechanical, and geometric Item Group SEM P value
properties of tibia obtained from
42-day-old broilers in the control 0 Suppl Cu25% Cu25% + phyt
group and supplemented with Cu (n = 10) (n = 10) (n = 10)
in 25% of daily demand
dependently on phytase Bone general properties
supplementation Bone weight (g) 19.4a 20.1a 26.2b 0.9 P < 0.001
Bone length (mm) 110a 110a 115b 1 P < 0.001
Bone weight/bone length (g mm−1) 0.176a 0.182a 0.228b 0.01 P < 0.001
Bone geometrical properties
Horizontal internal diameter h (mm) 4.41a 5.08b 5.76c 0.20 P < 0.001
Horizontal external diameter H (mm) 6.83a 8.62b 11.18c 0.50 P < 0.001
Vertical internal diameter b (mm) 2.07a 4.46b 4.81b 0.33 P < 0.001
Vertical external diameter B (mm) 4.52a 7.44b 8.70c 0.49 P < 0.001
Cross section area A (mm2) 17.1a 32.6b 55.0c 4.5 P < 0.001
Mean relative wall thickness MRWT (–) 0.86a 0.71b 0.89a 0.04 P = 0.009
Cortical index CI (%) 44.3ab 40.9a 46.3b 1.1 P = 0.024
Midshaft volume (cm3) 0.76a 1.44b 2.53c 0.21 P < 0.001
Moment of inertia Ix (mm4) 30a 157b 348c 40 P < 0.001
Index of gyration Rg (mm) 1.31a 2.16b 2.45c 0.14 P < 0.001
Bone mechanical properties
Yield strength (N) 149a 158a 383b 29 P < 0.001
Ultimate strength (N) 239a 248a 549b 40 P < 0.001
Elastic stress (MPa) 133a 46b 61b 11 P < 0.001
Ultimate stress (MPa) 215a 69b 86b 18 P < 0.001
Bending moment (N·m) 1.64a 1.75a 4.41b 0.35 P < 0.001

Mean values in rows with different lowercase letters differ significantly at P < 0.05; data given are means
SEM standard error of the mean, 0 Suppl the control group without received Cu in premix, Cu25% the group
received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the group received Cu in 25% of daily demand
from Cu-Gly with phytase

groups with the most significant increase being ob- Furthermore, the addition of phytase to the Cu-poor diet in
served in the Cu25% group supplemented with Cu with- the Cu25% + phyt group resulted in an increase of the total and
out the addition of phytase (Fig. 3). all particular zones of the growth plate cartilage as compared to

Table 4 The
histomorphometrical parameters Item Group SEM P value
of trabeculae of cancellous bone
in tibia obtained from 42-day-old 0 Suppl Cu25% Cu25% + phyt
broilers in the control group and (n = 10) (n = 10) (n = 10)
supplemented with Cu in 25% of
daily demand dependently on BV/TV (%) 16.2a 18.2b 21.5b 0.08 P < 0.001
phytase supplementation
Tb.Th mean (μm) 33.8a 55.5b 53.4b 1.69 P = 0.023
Tb.Th max (μm) 117a 150b 148b 7.45 P < 0.001
Tb.Sp mean (μm) 186a 287b 132a 13.00 P < 0.001
Tb.Sp max (μm) 597ab 696b 381a 39.70 P = 0.012
Fd (–) 1.54b 1.46a 1.62c 0.016 P < 0.001
Tb.N (mm−1) 4.75a 4.07a 6.13b 0.62 P < 0.001

Mean values in rows with different lowercase letters differ significantly at P < 0.05; data given are means
SEM standard error of the mean, BV/TV relative bone volume, Tb.Th trabecular thickness, Tb.Sp trabecular
separation, Fd fractal dimension of trabecular bone, Tb.N trabecular number, 0 Suppl the control group without
received Cu in premix, Cu25% the group received Cu in 25% of daily demand from Cu-Gly, Cu25% + phyt the
group received Cu in 25% of daily demand from Cu-Gly with phytase
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
Fig. 3 The morphology of the articular cartilage and the growth plate of
tibia obtained from 42-day-old chickens treated with Cu in organic (Cu-
Gly) form dependently on phytase supplementation. Data given are
the control group and in zone I and zone III when compared to
the birds fed the Cu-poor diet without the phytase additive (Fig.
3). The Cu-poor diet without the phytase addition resulted in an
increase of the total, zone II, and zone IV thickness when com-
pared to the control 0 Suppl group (Fig. 3).
Proteoglycan Content in the Articular Cartilage
In all examined groups, the osteochondral junction was intact
and the surface of the cartilage was smooth without irregular-
ities according to Mankin’s semiquantitative scoring system
(Fig. 4). Proteoglycan staining with SO showed a lower pro-
teoglycan content (displaying a weaker staining pattern) in the
cartilage from the control group (Fig. 4a), while the chickens
treated with the Cu-Gly at the concentration of 25% of daily
requirement demonstrated a higher, but only moderate, stain-
ing pattern linked with a higher content of proteoglycans. The
concentration of proteoglycans in the Cu-Gly-supplemented
groups, irrespective of the phytase addition, exhibited a grad-
ual increase with the distance from the periphery of the carti-
lage to the end of zone I and loss of safranin-O staining from
the beginning of zone II (Fig. 4b, c). The most intensive
347
mean ± SD. a, b Values with different letters differ significantly at
P < 0.05. The description of the groups as in Fig. 1
staining pattern with safranin-O was observed around
chondrocytes (Fig. 4b, c). In addition, there was no evident
gradient in the safranin-O staining within the control chickens
fed the Cu-deprived diet and their articular cartilages had a
very poor red staining pattern when compared to the other Cu-
Gly-supplemented groups (Fig. 4).
Distribution of Thick and Thin Collagen Fibers
and Proteoglycans in the Articular Cartilage
The structural information obtained from the analysis of
fibrous components in the PSR-stained section revealed
a difference between large (mature red-orange) and thin
(immature green) collagen fibers. The supplementation
of Cu (irrespective of phytase addition) enhanced thin
(green) and decreased thick (red) fibers in the articular
cartilage resulting in greener radial fibers (Fig. 5).
Moreover, thin collagen fibers (green) were distinctly
discernible in the layer located near the calcified carti-
lage at the cartilage-bone interface (Fig. 5b, c). An op-
posite proportion was observed in the control birds.
348 Muszyński et al.

Fig. 4 The representative images

of safranin-O staining carried out
on formaldehyde-fixed sections
from the tibial articular cartilage
of 42-day-old chickens treated
with Cu in organic (Cu-Gly) form
dependently on phytase
supplementation. The cartilage
from the control group displayed
a very low proteoglycan content
(displaying weaker staining
pattern), while the chickens
treated with the Cu-Gly at the
concentration of 25% of daily
demand demonstrated higher but
not strong staining patterns linked
with a higher content of
proteoglycans. The most
intensive staining with safranin-O
was observed around
chondrocytes. Vertical section of
zone I and upper part of zone II of
the tibial articular cartilage from
the control group fed the Cu-
deficient control diet (a), the
Cu25% + phyt group (b), and the
Cu25% + phyt group (c).
Magnification ×200. The
description of the groups as in
Fig. 1

Hormonal Analysis
The results of the analysis of the growth hormone, leptin, and
bone turnover markers are presented in Fig. 6. It was shown
that the addition of phytase to the Cu-poor diet for Ross 308
broiler increased the concentration of osteocalcin compared to
the other groups, while the concentration of IGF-1 increased
only when compared to the control Suppl 0 group (Fig. 6).
The experimental diets did not influence the serum concentra-
tion of other examined hormones, namely, growth hormone
and leptin.
Discussion
Adequate Cu intake in poultry production is necessary to not
only reach genetically optimal growth but also to maintain
proper function of the skeletal system, which is an important
mineral source and which provides structural support for well-
muscled and fast-growing breeds [14, 25]. For this reason, the
overloading of long bones could result in locomotion problems
that have a negative impact on the performance and welfare of
the intensively reared animals and could adversely affect the
profitability of livestock production [34]. Additionally, to pre-
vent other health problems associated with rapid growth, the
broilers of the fast-growing breeds are fed restricted amounts of
feed, especially during the rearing period [35, 36], which can
lead to aggressive behavior around feeders [37]. The mecha-
nisms for long bone growth are similar across most animal
species, but the growth rate is the fastest in the proximal tibia
of domestic fowl [38]. This, in combination with the general
weakness of the leg and other leg abnormalities in intensively
reared poultry, can deprive the birds of locomotive freedom,
leading to suffering, discomfort, fear, and distress during rear-
ing [34]. The syndromes that cause lameness in the birds of a
flock can affect one or several musculoskeletal tissues of a
single bird or of the whole flock at the same time [39]. Weak
bones or tendons result in, e.g., dyschondroplasia,
osteochondrosis, or bone loss. Thus, the potential beneficial
effects of supplementation with trace elements in organic form
on bone health are being increasingly investigated [15, 28, 40].
Such studies are necessary because supplementation with che-
lated forms of trace elements is an attractive strategy deserving
further evaluation in poultry breeding as traditional inorganic
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with... 349

Fig. 5 The representative images

of PSR staining carried out on
formaldehyde-fixed sections from
the tibial articular cartilage of 42-
day-old chickens treated with Cu
in organic (Cu-Gly) form
dependently on phytase
supplementation. The large
collagen fibers are orange or red,
and the thick ones, including
reticular fibers, are green. Vertical
section of zone I of the tibial
articular cartilage from the control
group fed the Cu-deficient control
diet (a), the Cu25% + phyt group
(b), and the Cu25% + phyt group
(c). Magnification ×200. The
description of the groups as in
Fig. 1

mineral salts are often used at levels higher than the recom-
mended dosage in order to avoid trace mineral deficiency [28].
Nevertheless, studies concerning how the skeletal system will
adapt to a Cu-poor diet are still limited and further studies are
therefore required, especially given that phytase is studied
mainly in relation to the bioavailability of P [41].
Our study showed that phytase addition to broilers supple-
mented with 4 mg kg−1 of organic Cu (the Cu25% + phyt
group) increased the final body weight, growth rate, and per-
centage tibia ash as compared to the broilers whose diets were
devoid of copper (the control 0 Suppl group) or those without
added phytase (the Cu25% group). It was not supported by

Fig. 6 The serum concentration

of insulin-like growth factor 1
(IGF-1), growth hormone,
osteocalcin, and leptin in 42-day-
old chickens treated with Cu in
organic (Cu-Gly) form
dependently on phytase
supplementation. Data given are
mean ± SD. a, b Values with
different letters differ significantly
at P < 0.05. The description of the
groups as in Fig. 1
350
other studies performed on broiler chickens that were fed con-
trol Cu-deficient diets, where increase of body weight after Cu
supplementation was observed at lowered levels [4, 42].
Furthermore, Cu bone content increased in both Cu-
supplemented groups when compared to the control 0 Suppl
group. These findings differed from those presented by Bao
et al. [42], where supplementation with organic Cu at 50% of
the recommended dose had no effect on the total Cu content of
tibia when compared to the Cu-deficient control group. Our
results also did not support studies showing that dietary
phytase supplementation improved mineral availability (P,
Ca, Cu, Mg, Fe) or Ca retention [43]. Our experimental diets
containing Cu at 25% of the recommended dose increased Cu,
Ca, P, and ash content in bone irrespective of phytase addition,
while the serum Ca concentration was decreased by phytase
supplementation. There are a few studies showing no effect of
dietary phytase supplementation on selected minerals [44, 45].
Furthermore, although serum Ca concentration decreased af-
ter phytase supplementation when compared to the Ca25%
group and bone Ca and P content increased in both Cu-
supplemented groups, the plasma and the bone Ca/P ratios
remained unchanged. Probably, the dietary P and Ca concen-
tration, bird breed, and age impacted Ca retention after
phytase supplementation while having no effect in P retention.
Similar observation was made by Chung et al. [43].
Our broiler chickens from the Cu25% + phyt group had
longer and heavier tibiae with an increased bone weight/bone
length ratio. Different results were obtained in the other study
with the Cu-deficient chickens. Here, no difference was ob-
served in the body weight or in the basal morphology of tibia
between chickens fed the Cu-poor diet and of those from the
Cu-deficient control group [46]. There is also another study
conducted with Cu-Gly given to growing chickens throughout
a 6-week period which showed that the reduction of dietary
copper to 25% of the recommended level resulted in an in-
crease in bone’s weight but without changes to its length [15].
In our study, a significant alteration was noted in bone
geometry, which was dependent on phytase supplementation.
Our broilers from the Cu25% + phyt group had more mature
bones with a greater cross-sectional area of the midshaft which
resulted in the increase of its mechanical strength and in de-
creased stresses. There was a 129 and 121% increase in the
ultimate strength of the bones in birds supplemented with
phytase (Cu25% + phyt), compared to the control Cu-
deficient (0 Suppl) or Cu-poor diet without the addition of
phytase (Cu25%), respectively. Significantly increased value
in yield strength revealed an increase to the extent of elastic
deformation after phytase supplementation. However, al-
though bones of birds which form the Cu25% + phyt group
were deforming and breaking under significantly higher loads,
the reduced values of yield and ultimate stresses showed that
tibiae from both Cu-supplemented groups were subjected to
lower mechanical stresses before fractures as compared to the
Muszyński et al.
control 0 Suppl group. In conclusion, it can be assumed that
the supplementation of Cu-Gly, even at lowered levels, posi-
tively influenced bone metabolism resulting in more mature
bone and better mechanical strength in growing broilers. It is
in agreement with results from another study performed on
Cu-deficient chickens [4]. The bones of birds from the Cu-
deficient control group had lower tolerance to deformation
and were able to absorb less energy before fracture.
Moreover, they demonstrated a reduced range of plastic de-
formation. However, even the lowest level of supplementation
(2 ppm) was shown to be sufficient in preventing the bone
changes seen in the Cu-deficient birds [4]. It seems that the
amount of Cu needed to maintain the mechanical integrity of
bone is substantially less than that required for growth [4].
Another study showed that after supplementing the diet with
phytase, the tibia became stronger [41]. It is suggested that
phytase supplementation results in better mineralization by
improving dietary quality through the release of other trace
minerals [17]. However, in contrast to our study, diets de-
scribed contained higher level of phytase (600–1050
FTU kg−1) and different level of phosphorus [17, 41].
More detailed comparison of our findings with those from
other studies is somewhat complicated, as no other studies spe-
cifically into the effects of dietary phytase supplementation in
Cu-poor diets are available. Our study has shown improvement
of the bone development in all Cu-Gly-treated broilers.
However, the reserve (I), proliferative (II), and hypertrophic
(III) zones in the tibia’s growth plate cartilage of the Cu25% +
phyt chickens were wider when compared to other birds. The
histological variation in birds with Cu deficiency suggested a
defective or disorganized proliferation and mineralization within
the analyzed zones of the growth plate. Thus, decreased bone
mechanical resistance observed in the control 0 Suppl group
could be linked with a failure in the mineral deposition within
the extracellular matrix of the bone. Furthermore, the chickens
fed the control 0 Suppl diet showed strongly osteoporotic can-
cellous bone. The widened reserve and hypertrophy zones ob-
served in the Cu25% + phyt group could be due to the increased
number of differentiating chondrocytes that resulted in the accu-
mulation of cells within particular zones after Cu and phytase
supplementation. Also, an increase in bone ash mineral content
and the trabecular number relating to the supplementation of
phytase and Cu-Gly were observed in the present study. Thus,
it is reasonable to speculate that the association of osteocalcin
with an increased cell proliferation and maturation in the growth
plate of our phytase-treated chickens mediated by the increase of
IGF-1 concentration could be the main cause of the improve-
ment of bone development. The results obtained in the current
study also indicate that the positive effects of phytase inclusion in
the Cu-Gly diet on growth and development were mediated by
improved activity of the somatotropic axis, as bone and cartilage
homeostasis is regulated by GH acting via IGF-1 stimulator of
the proliferation which was increased by 37% in the Cu25% +
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
phyt group. Some studies suggest an important role of nutrition
in the regulation of serum hormones like growth hormone, or
IGF-1, one of somatomedins [47]. Nevertheless, the mechanism
for exogenous enzymes—like phytase—on hormone regulation
is problematic and requires further examination. Currently, there
are a lack of studies relating to phytase supplementation and the
somatotropic axis. However, the study on dietary supplementa-
tion with the enzyme xylanase reported an increase in the con-
centration of blood IGF-1 in 21-day-old broilers. This indicates
that enhanced digestion and absorption of nutrients caused by
enzyme supplementation has effects on blood hormone concen-
tration [48].
The non-collagenous proteins contribute to a variety of
functions within the bone such as matrix stabilization, cal-
cification, and other metabolic regulatory activities.
Approximately 10% of different non-collagenous proteins
are expressed by osteoblasts and chondrocytes, and expres-
sion of these proteins is considered to be a specific marker
[49, 50]. Neither the proteoglycan content nor the alter-
ation in collagenous fibers in the articular cartilage was
reported in broiler chickens after nutritional modification
with phytase supplementation. Based on the obtained re-
sults, it can be concluded that both the presence of Cu in
the diet and the phytase inclusion significantly enhanced
the content of proteoglycans in the articular cartilage (Fig.
4). The phytase addition could exert a protective effect on
chickens’ articular cartilage in which degenerative changes
will occur much later when animals are fed the Cu-
deprived or Cu-poor diet, as was shown in the morphom-
etry and the distribution of proteoglycans (Fig. 4). The
phytase inclusion improved the morphology of the articu-
lar cartilage, and its beneficial effect was particularly man-
ifest in the widening of the superficial (I) zone. This
modification can positively alter the load distribution in
the joint and improve the elasticity of the articular carti-
lage, protecting it against the degradation in well-muscled
and fast-growing broilers. Furthermore, the increase of the
thickness of the transitional (II) zone can be equally ben-
eficial, as it can also improve the transfer and distribution
of the load through the joint. It can have other functional
consequences, as the physical properties of articular carti-
lage are determined by the diversity in the components of
the matrix and the difference in the quantitative mutual
relations of collagen fibers. The content of proteoglycans
plays an essential role in the destabilization of the colla-
gen network [49]. The proportion of thin and thick fibers
should give additional structural information about the in-
fluence of phytase addition on collagen synthesis in the
articular cartilage (Fig. 5). The fine (green) collagen fibers
might indicate an ongoing process of collagen synthesis
occurring after Cu supplementation, the intensity of which
was phytase-dependent (Fig. 5). Collagen is a major com-
ponent of the extracellular matrix of many tissues, and its
351
metabolism is directly associated with many physiological
processes of biological adaptation. Moreover, the bone or-
ganic matrix contributes to bone elasticity and changes in
the composition and spatial arrangement of soft bone tis-
sues may also be responsible for the improved mechanical
strength of bones observed in the experimented Cu-
supplemented groups [25].
The present study also revealed many side effects of the Cu-
deficient diet, e.g., very thin articular cartilage, especially the
shortening of the superficial (I) zone. It can result in accelerated
degradation of the articular cartilage linked with the loss of its
elasticity causing difficulties in movement. Furthermore, it can
provoke irreversible deformation caused by the impact of the
load during movement. Finally, the diet devoid of Cu supple-
mentation resulted in very low proteoglycan content in the
articular cartilage (Fig. 4). Proteoglycans also provide stability
to the articular cartilage, and the degradation of proteoglycans
entails destabilization of the collagen network.
Conclusions
To the best our knowledge, this is the first study that has
examined both the mechanical properties of bone as well as
the histomorphometry of cancellous bone and hyaline carti-
lage in chickens fed a diet low or deficient in copper but also a
diet low in copper in relation to phytase supplementation.
Irrespective of the phytase presence, the organic form of cop-
per can lead to changes within the articular cartilage, as was
indicated by the morphological analysis and the proteoglycan
content. Phytase supplementation showed beneficial effects
on differentiation in chicken growth plate chondrocytes, there-
by sustaining their proliferative state and maintaining their
sensitivity to growth factors, such as IGF-1. Furthermore,
the phytase addition influenced the trabecular architecture of
the tibia with subsequent improvement of its growth and de-
velopment. However, the response of the skeletal system to
phytase supplementation could differ among avian species
due to differences in physiology of the digestive tract and
gut microflora.
Acknowledgements The authors wish to thank ARKOP Sp. z o.o.
(Bukowno, Poland) for providing the necessary organically complexed
copper (GLYSTAR FORTE Cu) used for the purposes of this experiment.
This work was financially supported by the project NN 311543540 from
the Ministry of Science and Higher Education, Poland.
Compliance with Ethical Standards The experimental procedures
used throughout this study were approved by the Local Ethics
Committee on Animal Experimentation of the University of Life
Sciences of Lublin, Poland.
Conflict of Interest The authors declare that they have no conflict of
interest.
352
Open Access This article is distributed under the terms of the Creative
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creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
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priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
1. Hunt CD (1998) Copper and boron as examples of dietary trace
elements important in bone development and disease. Curr Opin
Orthop 9:28–36
2. Tomaszewska E, Dobrowolski P, Kwiecień M, Winiarska-Mieczan
A, Tomczyk A, Muszyński S (2017) The influence of the dietary
cu-glycine complex on the histomorphology of cancellous bone,
articular cartilage, and growth plate as well as bone mechanical
and geometric parameters is dose dependent. Biol Trace Elem Res
178:54–63. doi:10.1007/s12011-016-0894-x
3. Rodríguez JP, Ríos S, González M (2002) Modulation of the pro-
liferation and differentiation of human mesenchymal stem cells by
copper. J Cell Biochem 85:92–100. doi:10.1002/jcb.10111
4. Opsahl W, Zeronian H, Ellison M, Lewis D, Rucker RB, Riggins
RS (1982) Role of copper in collagen cross-linking and its influence
on selected mechanical properties of chick bone and tendon. J Nutr
112:708–716
5. Hong HH, Pischon N, Santana RB, Palamakumbura AH, Chase
HB, Gantz D, Guo Y, Uzel MI, Ma D, Trackman PC (2004) A role
for lysyl oxidase regulation in the control of normal collagen depo-
sition in differetnting osteoblast cultures. J Cell Physiol 200:53–62.
doi:10.1002/jcp.10476
6. Tomaszewska E, Dobrowolski P, Kwiecień M (2016) Alterations in
intestinal and liver histomorphology, and basal hematological and
biochemical parameters in relation to different sources of dietary
copper in adult rats. Ann Anim Sci. doi:10.1515/aoas-2016–0056
7. Yenice E, Mızrak C, Gültekin M, Atik Z, Tunca M (2015) Effects of
organic and inorganic forms of manganese, zinc, copper, and chro-
mium on bioavailability of these minerals and calcium in late-phase
laying hens. Biol Trace Elem Res 167:300–307. doi:10.1007/
s12011-015-0313-8
8. Scott A, Vadalasetty KP, Sawosz E, Łukasiewicz M, Vadalasetty
RKP, Jaworski S, Chwalibog A (2016) Effect of copper nanoparti-
cles and copper sulphate on metabolic rate and development of
broiler embryos. Anim Feed Sci Technol 220:151–158. doi:10.
1016/j.anifeedsci.2016.08.009
9. Banks KM, Thompson KL, Rush JK, Applegate TJ (2004) Effects
of copper source on phosphorus retention in broiler chicks and
laying hens. Poult Sci 83:990–996
10. National Research Council (1994) Nutrient requirements of poultry,
9th edn. National Academic Press, Washington. doi:10.17226/2114
11. Bao YM, Choct M, Iji PA, Bruerton K (2007) Effect of organically
complexed copper, iron, manganese and zinc on broiler perfor-
mance, mineral excretion and accumulation in tissues. J Appl
Poult Res 16:448–455. doi:10.1093/japr/16.3.448
12. Tomaszewska E, Dobrowolski P, Kwiecień M, Burmańczuk N,
Badzian B, Szymańczyk S, Kurlak P (2014) Alterations of liver
histomorphology in relation to copper supplementation in inorganic
and organic form in growing rats. Bull Vet Inst Pulawy 58:479–486.
doi:10.2478/bvip-2014-0073
13. Tomaszewska E, Dobrowolski P, Kwiecień M (2016) Intestinal
alterations, basal hematology and biochemical parameters in ado-
lescent rats fed different sources of dietary copper. Biol Trace Elem
Res 171:185–191. doi:10.1007/s12011-015-0522-1
Muszyński et al.
14. Świątkiewicz S, Koreleski J, Zhong DQ (2001) The bioavailability
of zinc from inorganic and organic sources in broiler chickens as
affected by addition of phytase. J Anim Feed Sci 10:317–328
15. Kwiecień M, Winiarska-Mieczan A, Zawiślak K, Sroka S (2014)
Effect of copper glycinate chelate on biomechanical, morphometric
and chemical properties of chicken femur. Ann Anim Sci 14:127–
139. doi:10.2478/aoas-2013-0085
16. Sebastian S, Touchburn SP, Chavez ER (1998) Implications of
phytic acid and supplemental microbial phytase in poultry nutrition:
a review. World Poult Sci J 54:27–47. doi:10.1079/WPS19980003
17. Aoyagi S, Baker DH (1995) Effect of microbial phytase and 1,25-
dihydroxycholecalciferol on dietary copper utilization in chicks.
Poult Sci 74:121–126
18. European Commission (2003) European Communities Directive
no. 1334/2003 of the European Parliament and the Council. OJ
L187:11–15
19. Aviagen (2014) Ross 308 broiler nutrition specification. Aviagen
Incorporated Publishing, Huntsville
20. AOAC (2000) Official methods of analysis of AOAC
INTERNATIONAL, 17th edn. AOAC International, Gaithersburg
21. Frühbeck G, Alonso R, Marzo F, Santidrián S (1995) A modified
method for the indirect quantitative analysis of phytate in food-
stuffs. Anal Biochem 225:206–212. doi:10.1006/abio.1995.1145
22. Žilić SM, Božović IN, Savić S, Šobajić S (2006) Heat pro-
cessing of soybean kernel and its effect on lysine availability
and protein solubility. Cent Eur J Biol 4:572–583. doi:10.
2478/s11535-006-0039-x
23. Tomaszewska E, Kwiecień M, Dobrowolski P, Klebaniuk R,
Muszyński S, Olcha M, Blicharski T, Grela ER (2016) Dose-
dependent effects of probiotic supplementation on bone character-
istic and mineralization in female turkeys. Anim Prod Sci. doi:10.
1071/AN16289
24. Ferretti JL, Capozza RF, Mondelo N, Montuori E, Zanchetta JR
(1993) Determination of femur structural properties by geometric
and material variables as a function of body weight in rats. Evidence
of sexual dimorphism. Bone 14:265–270
25. Muszyński S, Kwiecień M, Tomaszewska E, Świetlicka I,
Dobrowolski P, Kasperek K, Jeżewska-Witkowska G (2017)
Effect of caponization on performance and quality characteristics
of long bones in Polbar chickens. Poult Sci 96:491–500. doi:10.
3382/ps/pew301
26. Tomaszewska E, Kwiecień M, Muszyński S, Dobrowolski P,
Kasperek K, Blicharski T, Jeżewska-Witkowska G, Grela ER
(2017) Long bone characteristics are altered by caponization and
breed different. Br Poult Sci. doi:10.1080/00071668.2017.1280770
27. Dobrowolski P, Tomaszewska E, Kurlak P, Pierzynowski SG
(2016) Dietary 2-oxoglutarate mitigates gastrectomy–evoked struc-
tural changes in cartilage of female rats. Exp Biol Med 241:14–24.
doi:10.1177/1535370215595466
28. Tomaszewska E, Dobrowolski P, Kwiecień M, Wawrzyniak A,
Burmańczuk N (2016) Comparison of the effect of a standard in-
clusion level of inorganic zinc to organic form at lowered level on
bone development in growing male Ross broiler chickens. Ann
Anim Sci 16:507–519. doi:10.1515/aoas-2015-0087
29. Bobinac D, Spanjol J, Zoricic S, Maric I (2003) Changes in articular
cartilage and subchondral bone histomorphometry in osteoarthritic
knee joints in humans. Bone 32:284–290
30. Tomaszewska E, Dobrowolski P, Winiarska-Mieczan A,
Kwiecień M, Tomczyk A, Muszyński S (2017) The effect
of tannic acid on the bone tissue of adult male Wistar rats
exposed to cadmium and lead. Exp Toxicol Pathol 69:131–
141. doi:10.1016/j.etp.2016.12.003
31. Tomaszewska E, Dobrowolski P, Siwicki AK (2012) Maternal
treatment with dexamethasone at minimal therapeutic doses inhibits
neonatal bone development in a gender-dependent manner. Livest
Sci 146:175–182. doi:10.1016/j.livsci.2012.03.008
Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with...
32. Suvara SK, Layton C, Bancroft JD (2013) Bancroft’s theory and
practice of histological techniques, 7th edn. Churchill Livingstone
Elsevier, Oxford
33. Rich L, Whittaker P (2005) Collagen and picrosirius red staining: a
polarized light assessment of fibrillar hue and spatial distribution.
Braz J Morphol Sci 22:97–104
34. Mendes AS, Paixão SJ, Sikorski RR, Bonamigo DV, Morello MG,
Ponzoni RAR (2016) Photogrammetry: a non-invasive and objec-
tive method for detecting locomotion problems in broiler chickens.
Braz J Poult Sci 18:573–580. doi:10.1590/1806-9061-2015-007
35. van Krimpen MM, de Jong IC (2014) Impact of nutrition on welfare
aspects of broiler breeder flocks. World Poult Sci J 70:139–150.
doi:10.1017/S0043933914000129
36. De Jong IC, Jones B (2006) Feed restriction and welfare in domes-
tic birds. In: Bels V (ed) Feeding in domestic vertebrates. CABI,
Wallingford, pp 120–135
37. Hocking PM, Jones EKM (2006) On-farm assessment of environ-
mental enrichment for broiler breeders. Br Poult Sci 47(4):418–425.
doi:10.1080/00071660600825074
38. Kirkwood JK, Spratt DMJ, Duignan PJ, Kember NF (1989)
Patterns of cell-proliferation and growth-rate in limb bones of the
domestic-fowl (Gallus-domesticus). Res Vet Sci 47:139–147
39. Mench J (2004) Lameness. In: Weeks C, Butterworth A (eds)
Measuring and auditing broiler welfare. CABI, Wallingford, pp
3–17
40. Tomaszewska E, Muszyński S, Dobrowolski P, Kwiecień M,
Winiarska-Mieczan A, Świetlicka I, Wawrzyniak A (2017) Effect
of zinc level and source (zinc oxide vs zinc glycine) on bone me-
chanical and geometric parameters, and histomorphology in male
Ross 308 broiler chicken. Braz J Poult Sci 19:159–170. doi:10.
1590/1806-9061-2016-0285
41. Qian H, Veit HP, Kornegay ET, Ravindran V, Denbow DM (1996)
Effects of supplemental phytase and phosphorus on histological and
other tibial bone characteristics and performances of broilers fed
semi-purified diets. Poult Sci 75:618–626
42. Bao YM, Choct M, Iji PA, Bruerton K (2010) Trace mineral inter-
actions in broiler chicken diets. Br Poult Sci 51:109–117. doi:10.
1080/00071660903571904
353
43. Chung TK, Rutherfurd SM, Thomas DV, Moughan PJ (2013)
Effect of two microbial phytases on mineral availability and reten-
tion and bone mineral density in low-phosphorus diets for broilers.
Br Poult Sci 54:362–373. doi:10.1080/00071668.2013.783902
44. Rutherfurd SM, Chung TK, Thomas DV, Zou ML, Moughan PJ
(2012) Effect of a novel phytase on growth performance, apparent
metabolizable energy, and the availability of minerals and amino
acids in a low-phosphorus corn-soybean meal diet for broilers.
Poult Sci 91:1118–1127. doi:10.3382/ps.2011-01702
45. Um JS, Lim HS, Ahn SH, PaikK IK (2000) Effects of microbial
phytase supplementation to low-phosphorus diets on the perfor-
mance and utilization of nutrients in broiler chickens. Asian
Australas J Anim Sci 13:824–829. doi:10.5713/ajas.2000.824
46. Riggins RS, Cartwright AG, Rucker RB (1979) Viscoelastic prop-
erties of copper deficient chick bone. J Biomech 12:197–203
47. Hajati H, Rezaei M, Sayyahzadeh H (2009) The effects of
enzyme supplementation on performance, carcass character-
istics and some blood parameters of broilers fed on corn-
soybean meal-wheat diets. Int J Poult Sci 8:1199–1205.
doi:10.3923/ijps.2009.1199.1205
48. Gao F, Jiang Y, Zhou GH, Han ZK (2008) The effects of xylanase
supplementation on performance, characteristics of the gastrointes-
tinal tract, blood parameters and gut microflora in broilers fed on
wheat-based diets. Anim Feed Sci Technol 142:173–184. doi:10.
1016/j.anifeedsci.2007.07.008
49. Tomaszewska E, Dobrowolski P, Bieńko M, Prost Ł, Szymańczyk
S, Zdybel A (2015) Effects of 2-oxoglutaric acid on bone mor-
phometry, densitometry, mechanics, and immunohistochemistry in
9-month-old boars with prenatal dexamethasone-induced
osteopenia. Connect Tissue Res 56:483–492. doi:10.3109/
03008207.2015.1069822
50. Tomaszewska E, Dobrowolski P, Prost Ł, Hułas-Stasiak M,
Muszyński S, Blicharski T (2016) The effect of supplementation
of glutamine precursor on the growth plate, articular cartilage and
cancellous bone in fundectomy-induced osteopenic bone. J Vet
Med Sci 78:563–571. doi:10.1292/jvms.15-0385